The present invention relates to a method for determining the amount of ammonia in a body fluid and other samples and to a kit containing the reagents used to perform the method.
The determination of blood ammonia is important in the clinical diagnosis of a number of pathological conditions Elevated ammonia levels are associated with liver diseases, hepatic coma, Reye's syndrome, cardiac failure, and erythroblastosis fetalis. Monitoring of ammonia levels is also useful in the clinical management of patients who have undergone liver transplantation and hyperalimentation.
Determination of levels of ammonia in samples is also done in various industries ranging from the chemical to the food industries. Samples of chemicals, raw materials such as fertilizers and foodstuffs such as bakery goods or fruit juices are regularly analyzed.
Previous methods for the clinical determination of ammonia generally involved titrimetric or photometric analysis of ammonia following the conversion of ammonium ions to volatile form. Difficulties were encountered when systems contained volatile components other than ammonia. In addition, the assay conditions under which ammonia was determined could promote deamidation and deamination reactions, producing inaccurate results. For example, colorimetric determination of ammonia by the Berthelot reaction requires the deproteinization of the sample prior to analysis. Deproteinization, a process carried out at acid pH, can produce ammonia in situ, giving an erroneously high ammonia value.
The introduction of enzymatic methods for the determination of ammonia obviated the disadvantages described above. [see Scand. J. Clin. Lab. Invest., 16 (1964)443]. Such enzymatic systems utilized excess alpha-ketoqlutarate, nicotinamide adenine dinucleotide (NADH), and L-qlutamic dehydrogenase (GLDH). In such an enzymatic system, ammonia is contacted with alpha ketoqlutarate to form qlutamate, with concommitant oxidation of NADH to NAD. The decrease in NADH is directly related to the concentration of ammonia in the sample, and can be conveniently measured by determining the decrease in absorbance of NADH at 340nm.
In the clinical chemistry laboratory, early enzymatic methods utilized acid deproteinized whole blood as samples As described above, such deproteinization can produce an erroneously high ammonia value, affecting the accuracy of the determination. Mondzac et al. [see J. Lab. Clin. Med. 66 (1965)526] described an improved GLDH method which utilized heparinized plasma instead of deproteinized samples. A disadvantage of this method is that a true endpoint does not appear to exist, because other plasma components, such as enzymes, react with NADH. To circumvent this, protracted pre incubation periods of 15-20 minutes were utilized to pre-react these crossreactinq materials. However, such pre-incubation did not totally eliminate these crossreactions.
U.S. Pat. No. 3,929,581 describes an enzymatic method which is an improvement over Mondzac and which is indicated to be free from the disadvantages of earlier procedures. The '581 patent teaches use of non-deproteinized plasma as the sample. The sample is contacted with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and GLDH. In addition, adenosine diphosphate (ADP) is included to stabilize GLDH and accelerate the enzymatic reaction. The patentee reports that the assay takes approximately 5 minutes to perform, which is partially due to the fact that no pre-incubation period is needed. These factors combine to yield an assay essentially free of crossreactions and is therefore specific for ammonia.
The patentee argued that the use of NADPH in the place of NADH was not an obvious substitution of one nicotinamide adenine dinucleotide compound for another. Because of the cross reactivity problems seen with the use of NADH, the same were expected to occur with NADPH. It was also taught that GLDH reacted with NADH much more quickly than with NADPH, and that more undesired slow reaction would occur with the NADPH. However, neither of these problems arose with the method of '581.
An objective of this invention is to provide an alternative testing method for blood ammonia levels without the need for deproteinization and without the use of NADH. Another objective is the development of a blood ammonia assay that can be performed rapidly enough to be used on an automated diagnostic system. In particular, another objective of this invention is to provide for reagent test kits that can be used in automated diagnostic systems to test for blood ammonia.